The cancer associated membrane protein HER-2 is a member of the EGFR family of proteins. This particular protein has shown promise as an immunogen in active specific immunotherapy of certain cancers, notably breast cancer and colorectal cancer.
The assignee of the present patent application has previously filed patent applications relating to active vaccination against the HER-2 antigen, cf. WO 00/20027 which is hereby incorporated by reference herein. Further research in this field has now identified preferred HER-2 variants for such vaccines, but a general problem in protein chemistry is to devise improved means for obtaining satisfactory yields of recombinant protein with a high degree of purity.
Immobilized metal ion affinity chromatography (IMAC) was first introduced by Porath (Porath, J., J. Carlsson, I. Olsson, G. Belfrage [1975] Nature 258:598-599.) under the term metal chelate chromatography and has been previously reviewed in several articles (Porath, J. [1992] Protein Purification and Expression 3:263-281; and articles cited therein). The IMAC purification process is based on the employment of a chelating matrix loaded with soft metal ions such as Cu2+ and Ni2+. Electron-donating groups on the surface of proteins, especially the imidazole side chain of histidine, can bind to the non-coordinated sites of the loaded metal. The interaction between the electron donor group with the metal can be made reversible by lowering the pH or by displacement with imidazole. Thus, a protein possessing electron-donating groups such as histidine can be purified by reversible metal complex/protein interactions.
In 1991, Ford et al. (Ford, C., I. Suominen, C. Glatz [1991] Protein Expression and Purification 2:95-107) described protein purification using IMAC technology (Ni-NTA ligand) as applied to recombinant proteins having tails with histidine residues (polyhistidine recombinant proteins, “His-tagged proteins”). This method takes advantage of the fact that two or more histidine residues can cooperate to form very strong metal ion complexes.
Numerous variations of this technology exists, where the histidine residues are attached as “tags” to the relevant recombinant protein in various combinations, e.g. including recognition sites for specific proteases so that the his tag can be subsequently removed enzymatically.
Expression of proteins in insect cells require the use of various specialised culture media and also entails contamination of the recombinant protein with various insect cell derived constituents that are not found in bacteria, fungi and mammalian cells. Purification schemes devised for recombinant proteins produced in bacteria, fungi, or mammalian cells are therefore not necessarily the optimum choice when a protein produced in insect cells will need to be purified.
There is therefore a continuing need for improvements in protein purification in order to obtain pharmaceutical grade protein derived from recombinant production in insect cells.